Wnt/β-catenin signaling in the development and therapeutic resistance of non-small cell lung cancer

Wnt/β-catenin signaling is a critical pathway that influences development and therapeutic response of non-small cell lung cancer (NSCLC). In recent years, many Wnt regulators, including proteins, miRNAs, lncRNAs, and circRNAs, have been found to promote or inhibit signaling by acting on Wnt proteins, receptors, signal transducers and transcriptional effectors. The identification of these regulators and their underlying molecular mechanisms provides important implications for how to target this pathway therapeutically. In this review, we summarize recent studies of Wnt regulators in the development and therapeutic response of NSCLC. Supplementary Information The online version contains supplementary material available at 10.1186/s12967-024-05380-8.


Introduction
Lung cancer is one of the leading causes of cancer death worldwide, of which 80% to 85% is non-small cell lung cancer (NSCLC).Lung adenocarcinoma (LUAD) accounts for approximately 85% of NSCLC diagnoses, with lung squamous cell cancer (LUSC) accounting for approximately 15%, based on histological classification [1].The 5-year survival rate for NSCLC is only 26.5% because the disease is usually metastatic at diagnosis.Metastatic NSCLC is generally incurable, as it almost always develops therapeutic resistance after an initial response [2].
The Wingless/integrase-1 (Wnt) family is a type of secreted glycoproteins which interacts with transmembrane receptors and contributes to the development and differentiation of multiple organs, including lung [3].Wnt family proteins, of which there are 19 in humans, function as ligands to conduct a signal from the cell surface through the cytoplasm to the nucleus, thereby regulating expression of a coordinated sets of genes involved in multiple biological processes.Based on whether it relies on β-catenin for transcription activation, Wnt signaling pathways can be divided into the canonical pathway, namely Wnt/β-catenin signaling pathway, and non-canonical pathways, including Wnt/PCP pathway and Wnt/Ca 2+ pathway [4].Abnormal alterations of the Wnt/β-catenin pathway by its regulators contribute to the development and therapeutic responses of NSCLC [5].
β-catenin functions in a dual role, either as the most important nuclear effector of Wnt/β-catenin signaling, or as a cytoskeletal junction protein that maintains cell adhesion, which is critical for cadherin-based adherens junctions (AJs).These dual functions are carried out based on the transcriptional pool and the adhesive pool of β-catenin [6].In the transcriptional pool, Wnt ligands initiate a Wnt/β-catenin signaling cascade, which involves the translocation of β-catenin from cytoplasm to nucleus and activation of target genes via T cell factor (TCF)/lymphoid enhancer-binding factor (LEF) family of transcription factors (Fig. 1).In the absence of Wnt ligands, pathway signaling is inactivated by a "destruction complex" comprised of the tumor suppressor Adenomatous Polyposis Coli (APC), the scaffolding protein AXIN, casein kinase 1α (CK1α) and glycogen synthase kinase 3 β (GSK-3β) [7].Cytoplasmic β-catenin is sequestered in this destruction complex and sequentially phosphorylated by CK1α at Ser45 and GSK3β at Ser33/Ser37/Thr41, respectively [8].Phosphorylated β-catenin is then recognized by E3 ubiquitin ligase β-Trcp and ubiquitinated for proteasomal degradation [7].Without β-catenin in the nucleus, Groucho family transcription repressors bind to TCF/LEF transcription factors and inhibit the transcription of Wnt target genes.When present, Wnt ligands bind to the Frizzled (FZD) receptor family and a member of the low-density lipoprotein receptor-related protein (LRP) family, LRP5 or LRP6, to form FZD-LRP5/6 complexes.These complexes recruit the signal transducer Dishevelled (DVL) to the membrane for phosphorylation and oligomerization [9].Phosphorylated DVL recruits AXIN and inhibits its interaction with other components of the destruction complex, thereby preventing proteasomal degradation of β-catenin [10].Thus, the concentration of β-catenin will increase in the cytoplasm, translocating to the nucleus and forming a co-transcriptional complex with TCF/LEF, which activates the transcription of the downstream target genes which will regulate cell fate, migration, and tissue configuration [4].
In the adhesive pool, β-catenin acts as the core component of the AJs and regulates the aggregation of cadherin by directly binding to the cytoplasmic domain of E-cadherin and the actin-binding protein α-catenin, maintaining cell-cell junctions, tissue structural integrity, and homeostasis [11].The canonical function of the AJs is to initiate and stabilize cell-cell adhesion between neighboring cells and to modulate actin dynamics at the Fig. 1 An overview of Wnt/β-catenin signaling pathway.a In the absence of the Wnt signal, cytosolic β-catenin is phosphorylated by kinases CK1α and GSK3β with the help of scaffolding proteins AXIN and APC.Phosphorylation of β-catenin leads to its ubiquitylation and subsequent proteasomal degradation.b Wnt ligands bind FZD and LRP5/6 receptors on the cell surface.Subsequent phosphorylation of LRP5/6 and recruitment of signal transducers DVL and AXIN to the Wnt-bound receptors facilitate inhibition of GSK3β activity.This inhibition blocks phosphorylation and degradation of β-catenin, leading to β-catenin accumulation in the cytoplasm and translocation into the nucleus.In the nucleus, β-catenin interacts with TCF/LEF transcription factors to activate Wnt target genes cortical level, and dysfunctions of AJs contribute to cancer progression [12].
Epithelial-mesenchymal transition (EMT) comprises an essential biological process during which cells fail to maintain epithelial cell polarity and acquire a mesenchymal phenotype, thus facilitating invasion and metastasis.During the early phase of EMT, loss of apical-basal polarity is often the first event to be observed and can lead to the destabilization of adhesion complexes, including AJs at the lateral membrane [13].Wnt/β-catenin signaling is one of the most important pathways involved in the regulation of EMT.Wnt/β-catenin signaling exerts its effect on EMT through targeting and activating EMTtranscription factors SNAIL, SLUG, and TWIST which will regulate the expression of E-cadherin and N-cadherin.Wnt/β-catenin signaling can also impact EMT through AJs by other Wnt/β-catenin-targeted genes such as MMP7 and TIAM1 [14].
Wnt regulators influence Wnt/β-catenin signaling at both the transcriptional and translational level, with regulators identified that act on ligands, receptors, signal transducers and transcriptional effectors.These regulators might be proteins, microRNAs (miRNAs), long noncoding RNAs (lncRNAs), or circular RNAs (circRNAs) [5].miRNAs contain 20-25 nucleotides which repress translation of targeted mRNAs or target mRNA degradation [15].LncRNAs are RNA transcripts longer than 200 nucleotides, and most of them do not encode peptides.LncRNAs encompass natural antisense transcripts, overlapping transcripts and intronic transcripts, which regulate gene expression through a variety of different mechanisms, including acting as molecular scaffolds that 'guide' chromatin-modifying enzymes, competing endogenous RNAs (ceRNAs) that 'sponge' miRNAs or proteins, facilitating or inhibiting long-range chromatin interactions, or functioning through the act of transcription itself [16].CircRNAs are a class of single-stranded noncoding RNAs in circular form through non-canonical splicing or back-splicing manner.CircRNAs can serve as miRNA sponge in which circRNAs bind directly to the targeted miRNAs to inhibit miRNA activity, or affect alternative splicing through RNA-mediated interaction, or interact with RNA-binding proteins as protein scaffolds or antagonists [17].
Based on functional effect, Wnt regulators can be classified as positive and negative regulators.The upregulation of positive regulators and downregulation of negative regulators will promote the activation of Wnt/ β-catenin signaling pathway.Aberrant Wnt regulator expression and signaling have been identified in lung cancer cell lines, animal models, and human NSCLC tissues [18][19][20][21][22][23].Modulation of these regulators provide potential treatment strategies for patients with NSCLC, and many agents that suppress Wnt/β-catenin signaling also inhibit NSCLC cell lines [24,25].In this review, we mainly focus on the recent studies of regulators identified in Wnt/β-catenin signaling implicated in development and therapeutic responses of NSCLC.

Aberrant alterations of Wnt components in NSCLC
In humans, the complexity and specificity of Wnt signaling is achieved partially through 19 Wnt ligands [4].The aberrant expression of most Wnt ligands have been found to closely correlate to the occurrence and progression of NSCLC, and are the thus potential biomarkers and drug targets for the diagnosis, prognosis, and treatment of NSCLC [26].Overexpression of WNT2B, WNT3A and WNT5A has been found to associate with NSCLC [27,28] (Table 1).FZD family members are a type of seven-pass transmembrane receptor (FZD1-FZD10) that belong to atypical G protein-coupled receptors (GPCRs).Specifically, FZD2 expression was found to associate with the prognosis of LUAD [29], and promoter CpG methylation of FZD2 might be related to the prognosis of LUSC [30].Abnormal expression of many FZDs (FZD3, FZD8 and FZD9) is associated with the development of NSCLC [3].It has been observed that patients with early-stage NSCLC carrying the SNP rs10898563 in FZD4 showed a significant increase in recurrence and mortality risk [31], and FZD4 expression might be associated with the prognosis of LUAD [29].Knockdown of FZD8 by shRNA sensitized the lung cancer cells to chemotherapy [32].FZD10 methylation was found to possibly relate to the prognosis of patients with LUSC [30] (Table 1).
For LRP5/6 receptor, LRP5 expression has been shown to be decreased in LUSC [33].SNPs in LRP5 were found to associate with an higher risk of NSCLC (SNP rs3736228) and LUSC (SNP rs64843) [34].SNP rs10845498 on LRP6 is associated with a lower risk of LUSC, whereas LRP6 rs6488507 is associated with higher risk of NSCLC in tobacco smokers [35].For Dishevelled (DVL), upregulated expression of DVL1 and DVL3 was found in brain metastases from LUAD [36].Overexpression of DVL1 is associated with unfavorable prognosis of patients with NSCLC [37] (Table 1).
For components of the destruction complex, AXIN1 methylation was found to correlate with radiosensitivity of lung cancer cells and clinical features of NSCLC [38,39] (Table 1).Downregulation of AXIN1 expression was found in micropapillary-predominant LUAD, especially in cases with lymph node invasion, indicating diminished AXIN1 expression may affect the invasiveness of LUAD [40].The intronic AXIN2 1712 + 19 variant exhibited increased mortality in Indian LUAD patients with GG genotype [41], while the heterozygous (GT) genotype showed a decreased risk of mortality [42].AXIN2 148 C/T and 1365 C/T variants might be associated with reduced cancer susceptibility in Chinese NSCLC patients [43,44].Aberrant promoter methylation of AXIN2 was observed in NSCLC, and might be related to prognosis and histological subtyping of NSCLC [45].High expression of CSNK2A1, which encodes CK1α, is an independent prognostic factor of poor survival for NSCLC patients [46] (Table 1).APC and CTNNB1 mutations were also found in NSCLC (Fig. 2).In NSCLC, APC mutations are mostly loss-of-function truncating mutations which are evenly distributed across APC gene (Fig. 2a; Supplementary Table 1); CTNNB1 mutations are mostly gain-offunction point mutations that mainly concentrate on the GSK3β/CK1α phosphorylation sites (Fig. 2b; Supplementary Table 2).The mutations on phosphorylation sites prevent the phosphorylation of β-catenin and so escape from E3 ubiquitin ligase β-Trcp and subsequent proteasomal degradation, thus leading to the accumulation of β-catenin and elevated Wnt/β-catenin signaling [47].

The positive regulators of Wnt/β-catenin signaling
Many positive regulators have been identified which act on Wnt ligands, receptors, transducers, components of β-catenin destruction complex, and β-catenin.These regulators might be overexpressed, amplified, or mutated in NSCLC cells.

Wnt ligands
The expression of multiple Wnt ligands have been found to be upregulated in NSCLC, including WNT1, WNT2B, WNT3A and WNT5A (Table 2).WNT1 transcriptional expression was upregulated by PHF8, a histone demethylase.Higher PHF8 expression was found in NSCLC and correlated with poorer overall survival in NSCLC patients.Mechanistically, PHF8 increases WNT1 transcription by targeting the promoter region of WNT1 and so removing the histone markers there [48].WNT2B expression was upregulated by the RNA helicase DDX56 [49] and lncRNA RPPH1 [50].DDX56 overexpression TCF-4 The 10th exon partial deletion TCGA LUAD Better overall survival [258] was found in LUSC and negatively associated with recurrence-free survival in LUSC patients.DDX56 increased the transcription of the target gene WNT2B through the degradation of primary miR-378a [49].RPPH1 overexpression was negatively associated with disease progression and overall survival.Mechanistically, lncRNA RPPH1 promoted NSCLC progression through miR-326/WNT2B axis as WNT2B is a target gene of miR-326 [50].Another lncRNA, AL139294.1,promotes WNT5A expression and oncogenic activity through suppression of miR-204-5p [51].WNT3A expression was upregulated by PITX2 [52], ASPM [53], GOLPH3 [54], ALDOC [55] and FAIM2 [56] (Table 2).PITX2 binds directly to the promoter of WNT3A and upregulated its transcriptional expression.High PITX2 expression was found in LUAD and correlated with worse prognosis [52].GOLPH3 is a peripheral membrane protein localized to the trans-Golgi.High expression of GOLPH3 was found in NSCLC tissues and was associated with clinicopathologic characteristics.GOLPH3 interacts with CKAP4 and increases the secretion of exosomal WNT3A, leading to a cancer stem cell (CSC)-like phenotype and metastasis in NSCLC [54].WNT5A expression was found to be upregulated by PTS [57], circVAPA [58], E2F1 [59] and ATF4 [60] (Table 2).Higher PTS level was found in LUAD and correlated with late clinical stages and poor survival [57].circVAPA acted as a ceRNA to up-regulate WNT5A by sponging miR-876-5p and thus activating Wnt/β-catenin signaling [58].Intriguingly and perhaps paradoxically, WNT5A has also been reported to inhibit Wnt/β-catenin signaling in EGFR-mutant cells.In this scenario, E2F1mediated repression of WNT5A expression promotes brain metastasis EGFR-mutant NSCLC, and high expression of E2F1 was negatively correlated with the expression of WNT5A and associated with poor outcomes in NSCLC [59].

Wnt receptors
Many positive regulators act on Wnt receptors by multiple mechanisms in NSCLC.FZD1 expression is upregulated by LINC00942 in LUAD (Table 2).Higher expression of LINC00942 was found in LUAD tissues and associated with poorer survival.Mechanically, LINC00942 functioned as a ceRNA which targets miR-5006-5p and increases the expression of its direct target FZD1 [61].FZD4 expression was found upregulated  Circ_0017109 regulated FZD4 expression by targeting miR-671-5p and finally activated Wnt/β-catenin signaling [62].
The phosphorylation of LRP5/6 recruits AXIN and GSK3β to its phosphorylated sites, leading to the disassembly of β-catenin destruction complex.As a result, β-catenin accumulate in cytoplasm which finally translocate to the nucleus and enhance the transcription of targeted genes [63].LRP5/6 phosphorylation is upregulated by ENO1 [19] (Table 2), which is a metabolic enzyme involved in the synthesis of pyruvate.ENO1 also decreased GSK3β activity, inactivated the β-catenin destruction complex and ultimately upregulated β-catenin.Higher expression of ENO1 was found in metastatic lung cancer cell lines and patients, and associated with worse overall survival of patients with NSCLC [19].LRP6 can directly interact with TRIP13 [64] and NINJ1 [65] (Table 2).TRIP13 is an ATPase which is highly expressed in NSCLC, correlating with advanced tumor stage and poor patient survival.TRIP13 promotes NSCLC cell proliferation and invasion through activating Wnt/β-catenin signaling [64].NINJ1 is a 17-kDa homophilic cell adhesion molecule located in the cell membrane.NINJ1 overexpression was found to associate with poor prognosis in patients with NSCLC.Mechanistically, NINJ1 forms an assembly with LRP6 and FZD2, resulting in transcriptional upregulation of Wnt downstream target genes [65].CD248 inhibits the interaction between LRP6 and Wnt repressors IGFBP4 and LGALS3BP, increasing Wnt/β-catenin signaling in pericytes to promote angiogenesis and tumor growth in lung cancer [66].Ubiquitylation also participate into the regulation of LRP6.USP46 is a deubiquitylase which form complex with the catalytic USP46 and the WDR40-repeat proteins, WDR20 and UAF1.This complex increases the steady-state level of cell surface LRP6 and facilitates the assembly of LRP6 into signalosomes through the removal of sterically hindering ubiquitin chains.Alterations in USP46 mostly consisted of amplification and were commonly observed in LUSC [67].
The complex of nuclear β-catenin and TCF4 transcription factor was upregulated by nuclear E-cadherin [149], Pygo1 [150], FOXP3 [151] and TRIB3 [152] (Table 2).β-catenin/TCF4 interaction was abolished by E-cadherin and was correlated with its nuclear localization, and consequently decreased β-catenin/TCF4 transcriptional activity.Subsequently, nuclear E-cadherin was a negative regulator of Wnt/β-catenin-elicited promotion of lung CSC phenotype [149].FOXP3 can physically interact with TCF4 and β-catenin in the nucleus.High level of FOXP3 had a significant decrease in overall survival and recurrence free survival NSCLC patients [151].

The negative regulators of Wnt/β-catenin signaling
Many negative regulators have been identified which act on Wnt ligands, receptors, components of β-catenin destruction complex, and β-catenin.The expression of these regulators might be achieved by aberrant expression, mutation, methylation, and histone modifications in NSCLC cells.

Wnt/β-catenin signaling impacts therapeutic sensitivity and resistance of NSCLC
The abnormal activation of Wnt components and regulators influences response to several therapies for NSCLC, including targeted therapy, radiotherapy and chemotherapy.

Chemotherapy
Cisplatin (DDP) is the most widely used chemotherapeutic agent for NSCLC [220,221].DVL2 overexpression was found in DDP-resistant NSCLC A549 (A549/ DDP) cells compared to the parental A549 cells (Table 4).Inhibition of DVL2 resensitizes DDP-resistant NSCLC cells through downregulating Wnt/β-catenin signaling [222].RRM2 is a component of ribonucleotide reductase.Higher levels of RRM2 expression was found in A549/ DDP cells.Knockdown OF RRM2 promoted the sensitivity of A549/DDP cells to cisplatin through Wnt/β-catenin signaling pathway [223].TPX2 is a microtubule-related protein in mobile mitosis and spindle assembly [224].
Transmission of exosomal TPX2 promotes the resistance of NSCLC cells to docetaxel through increasing the protein level of β-catenin [225].Many miRNAs have been found to involve in chemotherapy of NSCLC.miR-32 and miR-548a were poorly expressed in DDP-resistant NSCLC, re-expression of miR-32 and miRNA-548a promotes the sensitivity of NSCLC cells to cisplatin by targeting ROBO1/β-catenin axis [226].miR-181c expression was upregulated in DDP-resistant NSCLC cells, and miR-181c negatively regulated WIF1 expression through directly binding to WIF1 (Table 4) [227].

Radiotherapy
Wnt/β-catenin signaling has been found to associate with radiotherapeutic sensitivity and resistance of NSCLC.WNT5A expression is often upregulated in radiationresistant NSCLC cells (Table 4).Mechanistic investigation indicated that altered WNT5A expression affects radiosensitivity of NSCLC via Wnt/β-catenin pathway [228].Disabled-2 (Dab2) is known as a tumor suppressor and Wnt pathway inhibitor.It has been found that promoter de-methylation of Dab2 gene enhances X-Ray irradiation sensitivity of NSCLC cells [229].Cancerassociated fibroblasts (CAFs), one main component of the tumor microenvironment, regulated DNA damage response of NSCLC cells following irradiation.
Mechanistically, CAFs up-regulate and stabilize c-Myc, leading to the transcription activation of HK2 kinase, a key rate-limiting enzyme in glycolysis by activating Wnt/ β-catenin pathway [230].Therefore, CAFs contribute to the radioresistance of NSCLC cells by promoting the glycolysis in a Wnt/β-catenin signaling-dependent manner.UBE2T has been found to promote NSCLC progression [231].Recently, it was found that UBE2T promotes radioresistance in NSCLC (Table 4).Mechanistically, UBE2T promotes EMT partially through Wnt/β-catenin signaling activation [232].Therefore, Wnt/β-catenin signaling might be a potential target for enhancing radiotherapy sensitivity.

Immunotherapy
Aberrant activation of Wnt/β-catenin signaling promotes the escape of cancer cells from immune surveillance, inhibits T-cell infiltration, and mediates the response to immunotherapy [233,234].It has been shown that WNT1 silences chemokine genes in dendritic cells and induces adaptive immune resistance in LUAD [235].Tumor β-catenin expression is associated with immune evasion in NSCLC with high tumor mutation burden [236].By bioinformatic analysis, DKK1 was identified as a candidate gene related to composition of tumor immune microenvironment and response to immunotherapy in LUAD patients [237].Therefore, Wnt/β-catenin pathway might be a potential mechanism involved in the regulation of response to immunotherapy.

Potential NSCLC treatments through suppression of Wnt/β-catenin signaling
Multiple small molecules exist which inhibit positive Wnt regulators, providing an avenue to suppress Wnt/ β-catenin signaling in NSCLC.Porcupine protein, a membrane bound O-acetyltransferase, regulates the biogenesis of Wnt ligands.The Porcupine inhibitor LGK-974 functions by binding to Porcupine and competing with acyl-CoA, thus blocking Wnt acetylation by Porcupine and inhibiting Wnt/β-catenin signaling [238] (Table 5).
LGK-974 modifies tumor-associated macrophages resulting in inhibition of NSCLC cells [239], with one Phase 1 study still active (NCT01351103).Similarly, NCT-80 is an Hsp90 inhibitor which upregulates the transcription of Wnt ligands through Akt-and ERK-mediated activation of STAT3 (Table 5).NCT-80 effectively overcomes acquired resistance to chemotherapy and EGFR targeting anticancer therapy by inducing apoptosis and inhibiting EMT [240].USP5 has also been found to be a positive regulator of Wnt/β-catenin signaling in NSCLC.Targeting USP5 with the small molecule WP1130 induced the degradation of β-catenin, and showed markedly inhibitory effects on tumor growth and metastasis [122].
Many chemicals can inhibit the development and therapeutic resistance of NSCLC by targeting Wnt/βcatenin signaling.Ethacrynic acid, a loop diuretic, suppresses EMT of A549 cells via blocking of NDP-induced Wnt signaling [245] (Table 5).IMU1003 is an atrarate derivative which dramatically decreased the emergence of osimertinib-resistant colonies through inhibiting the nuclear localization of β-catenin [246] (Table 5).
Antisense oligonucleotide (ASO) drugs have been reported to be effective at inhibiting tumor growth both in vitro and in vivo (Table 5) [253].LncRNA PKMYT1AR promotes CSC maintenance in NSCLC via activating Wnt signaling pathway.PKMYT1AR targeting ASO was found to dramatically inhibit tumor growth in vivo [99].
Nanoparticle formulations can improve the efficacy of existing drugs.Berberine, an isoquinoline alkaloid known for its anti-cancer and anti-inflammatory properties, shows low solubility and bioavailability (Table 5).The physiochemical functions of berberine can be largely improved by being encapsulated into liquid crystalline nanoparticles.Berberine liquid crystalline nanoparticles significantly suppresses the expression of β-catenin at both transcription and translation level [254].

Fig. 2
Fig. 2 APC and CTNNB1 mutations identified in NSCLC from GENIE datasets (GENIE 14.0-public, n = 26,473).a The recurrent truncating APC mutations (n ≥ 2) in NSCLC were shown on the schematic structure.The truncating mutations include nonsense mutations and frameshift mutations.b The recurrent CTNNB1 mutations (n ≥ 2) were shown on the schematic structure.β-catenin is sequestered in the destruction complex and sequentially phosphorylated by CK1α at Ser45 and GSK3β at Ser33/Ser37/Thr41, respectively.The truncating mutations were not included in this study

Table 1
Wnt components which have been reported to associate with NSCLC

Table 3
Negative Wnt regulators recently reported to involve in NSCLC

Table 4
Wnt regulators recently reported to associate with the therapeutic sensitivity and resistance of NSCLC CAFs cancer-associated fibroblasts, PFS progression-free survival

Table 5
Agents recently reported to inhibit the development and therapeutic response of NSCLC through regulating Wnt/β-catenin signalingConclusionsRecent identification of multiple Wnt regulators, and their dysregulation in NSCLC, emphasize the importance of Wnt/β-catenin signaling in NSCLC development and therapeutic response.These regulators act on Wnt ligands, receptors, signal transducers, and transcriptional effectors, as well as those well-known regulators.Dysregulation of these Wnt regulators can be either genetic or epigenetic, resulting in overexpression, underexpression, or gain of function and loss of function.Multiple circRNAs and micropeptides have been found to regulate Wnt/β-catenin signaling in NSCLC.Continued study of these regulators improves our understanding of NSCLC biology and may open avenues to novel therapies through the direct targeting of Wnt/β-catenin signaling.